Full blood count (FBC) is a diagnostic test that is used to measure cellular composition of blood. It may give information about the status of an immune system of a patient, about the ability of the blood to disseminate oxygen and/or about the ability of the blood to effectively clot. As such, it is a fundamental test that is often used as an initial “general purpose” diagnostic tool or as a more targeted monitoring solution. Examples of care cycles that include a full blood count as a monitoring tool include oncology, arthritis and Crohn's disease. As many as 300 million FBC tests are performed each year in the developed world.
FBC diagnostic parameters and their clinical indicators are summarized in Table 1 and Table 2 hereunder. These parameters are generated from several individual measurements, specifically a white blood cell (WBC) differential count, a red blood cell (RBC) count, a platelet count and a hemoglobin (Hb) measurement (see also FIG. 1).
TABLE 1FBC Clinical Parameters relating to the red blood cells.Clinical SignificanceDiagnostic parameterDetailsDecreaseIncreaseHemoglobin (Hb)Concentration of Hb inHemolytic anaemiaErythrocytosis:lysed whole bloodHaemorrhageCongenital heart(g/dl)Poor dietdiseaseBnone marrow failureChronic hypoxiaRenal disease(shortage of oxygen inNormal pregnancythe body).Rheumatoid/collagenSevere dehydrationvascular diseaseExcess RBCMultiple myelomaproduction by boneLeukaemiamarrowHodgkin diseaseRBC countNumber of RBCs perHaemorrhageErythrocytosis:mm3Poor dietReduced O2 capacityBone marrow failureof HaemoglobinRenal DiseaseExcess RBCproduction by bonemarrowHigh altitudeSevere dehydrationCongenital heartdiseaseMean CorpuscularAverage RBC volumeIron deficiency anaemiaVitamin B12Volume (MCV)ThalassemiadeficiencyFolic acid deficiencyChemotherapyLiver diseaseRBC distribution% variation from meanN/AVitamin B12width (RDW)RBC volumedeficiencyFolic acid deficiencyIron deficiencyanaemiaMean corpuscularAverage conc. of Hb inMicrocytic anemiaMacrocytic anemicHemoglobin (MCH)each RBC(small RBC and/or less(large RBC and/orHb)more Hb)Mean corpuscularAverage weight of HbIron deficiency anemiaIntravascularHemoglobinper RBCThalassemiahemolysis (free Hb inConcentrationblood)(MCHC)Hematocrit% (v/v) concentrationHb disorderPolycythemia veraof RBC's in wholeCirrhosis(excess RBCbloodHemolytic anaemiaproduction by boneHaemorrhagemarrowDietary deficiencyHigh altitudeBone marrow failureSevere dehydrationRenal diseaseCongenital heartNormal pregnancydiseaseRheumatoid/collagenvascular diseaseMultiple myelomaLeukemiaHodgkin disease
TABLE 2FBC Clinical Parameters relating to the white blood cells.Clinical SignificanceDiagnostic parameterDecreaseIncreaseTotal white blood cell (WBC)Bone marrow suppression dueViral, bacterial, fungal, orcountto chemotherapy, radiationparasitic infection.Therapy, leukaemia or disease-Cancer.modifying drugs.Response to certain medicationsPeripheral Blood Mononuclear Cells (PBMCs)Lymphocyte count (cells/mm3)SepsisIntracellular infection (viral orLeukaemiabacterial)ImmunodeficiencyLatter stages of an HIVinfectionDrug therapy (e.g.adrenocorticosteroids)Radiation therapyMonocyte count (cells/mm3)Drug therapy: PrednisoneViral infectionsParasitic infectionChronic inflammatory disordersTuberculosis (TB)GranulocytesNeutrophil count (cells/mm3)Overwhelming bacterialAcute bacterial infectioninfection (esp. in elderly)Inflammatory disorders (e.g.Viral infectionrheumatoid arthritis)Dietary deficiencyMyelecystic lukemiaAplastic anaemiaMetabolic disordersRadiation therapyTraumaDrug therapy: Myelotoxic drugsPhysical or emotional stress(as in chemotherapy)Eosinophil count (cells/mm3)Increased adrenosteroidAllergic reaction.productionAutoimmune disease.Parasitic infections.LeukaemiaBasophil count (cells/mm3)Myeloproliferative diseaseAcute allergic reactionLukemiaHyperthyroidismStress
Currently, large scale commercial laboratory instruments known as hematology analyzers are used to automatically perform all measurements that comprise the FBC. The high cost and complexity of these devices, coupled to the need for venous blood, means that they are mostly large scale, centralized facilities.
There is a clear clinical need for performing FBC in a near patient setting, particularly for applications that require a full blood count to monitor the progression and/or treatment of a disease. Microfluidic point of care devices have been developed which are capable of measuring individual components of the FBC. In that area, Hb measuring devices, WBC counters capable of performing a white blood cell differential and platelet count devices, devices which optically count and determine size of red blood cells are available.
For cell counting, current hematology analyzers typically employ electrical coulter counting and/or optical scattering methods to count and differentiate white cells and to count and determine size of the red blood cells and platelets.
At the moment only few examples of microfluidic coulter counter technologies exist. One example combines a coulter counter with a Hb measurement. Another example of counting cells is by flow-through impedance spectroscopy. This is a new flow cytometry analysis which is especially suited for a micro fluidic format. This technique is capable of differentiating between lymphocytes, monocytes and neutrophils in lysed blood, and of counting and sizing red blood cells and platelets.
The current “gold-standard” for Hb measurement is the photometric cyanmethaemoglobin (HbCN) method [see van, K. E. and W. G. Zijlstra, Standardization of hemoglobinometry II, The hemiglobincyanide method, Clin Chim Acta, 1961, 6, p. 38-44]. This method involves chemical lysis of the red blood cells and subsequent labelling of all the Hb that these cells release with a cyanide ion. The labels produce a defined absorption profile with a maximum at 540 nm. By measuring the optical absorption at 540 nm, the concentration of Hb can be determined. Furthermore, the high stability of HbCN means that it is easy to supply a calibration standard.
The most common red blood cell lysis/cyanide conversion reagent is known as Drabkin's reagent. Drabkin's reagent contains Potassium Cyanide, which is extremely toxic. This reagent only works for very large dilutions in whole blood (1:251), since red blood cell lysis relies on the low ionic strength of the reagent to induce osmotic shock. This large dilution causes an inherent imprecision in the method. Furthermore, to measure the optical absorption at 540 nm, very long optical path lengths of ˜1 cm are required. Finally, in some pathological samples, turbidity can lead to erroneously high absorption readings, which in turn will give rise to an incorrect Hb concentration.
To avoid the problems associated with toxicity and turbidity, many other optical means of measuring Hb have been developed. Examples of these will be described below.
A known point of care device uses sodium azide to convert the Hb to an azide-coordinated Hb derivative (azidemethemoglobin, HbN3). This method itself lends to short path length (0.1 mm) absorption spectroscopy, since dry reagents remove the need for dilution of the whole blood. Two absorbance readings are taken to determine the HbN3 concentration, i.e. one at the absorption maximum (565 nm) and one at 800 nm to correct for turbidity.
For the point of care WBC/Hb counter, a RBC lysis solution has been developed that preserves the WBCs while at the same time labeling the Hb molecule with imidazole. In a similar way as described above, the optical absorption of the imidazole labeled Hb species is measured at two wavelengths, i.e. one at the absorption peak and one to correct for turbidity and scattering effects for the white blood cells. The same solution may also be passed through a coulter counter to perform the cell count.
Another known lysis/Hb conversion reagent is based on sodium lauryl sulphate/sodium dodecyl sulphate (SLS/SDS). The SDS lyses all the blood cells and labels the Hb to get an SDS-coordinated derivative. Since SDS is a surfactant molecule, turbidity correction is not necessary and so a single absorption reading at 535 nm is taken to determine the Hb concentration. This method is designed for high dilutions of Hb, so the inherent imprecision present in the HbCN measurement is still present in the HbSDS one.
All the above described devices and techniques are capable of performing specific measurements from a finger-prick of blood. However, none of the above described devices and techniques are capable of measuring all parameters that are required for an FBC at ones. In other words, none of the devices and techniques described above are able to perform a complete FBC test at the point of care.